| Collagen is the main structural protein in organism.Natural collagen has good biological security and biocompatibility,was widely applicated in functional materials field.Compared with traditional mammal collagen,fish collagen source is much wider,safer and cost lower which made it a good substitute for mammal collagen.At present,extracting collagen from fish processing waste resources and processing it into all kinds of food and health care products has become the latest trends of fish processing and comprehensive utilization.But,processing fish collagen into biomedical materials research is still rare.Therefore,this study use a common fish species in the Yangtze river--Leiocassis longirostris skin as raw material,separate and purify fish collagen,and carried out the studies of macromolecular structure and biological properties for carnivorous fish collagen supplement relevant scientific information.On this basis,try to take advantage of the self-assembly molecular behavior of natural source fish collagen in vitro and the effect of centrifugal force to synthesis oriented collagen as tendon bionic biological materials.And carry out system research of oriented assembly optimization,material structure and material performance analysis for the application of fish collagen in the field of biomedical materials and providing technical support for the development of novel materials.Using Leiocassis longirostris skin as raw material,extract acid soluble collagen(ASC) with acid and enzyme soluble collagen(PSC) products with acid/enzyme.Adopt spectral analysis,SDS-page analysis and amino acid composition analysis to study molecular structure characteristics of Leiocassis longirostris collagen.Collagen enzyme stability performance and stability performance was analyzed by using DSC and in vitro enzyme degradation.Results showed that the ASC and PSC extracted were typical type Ⅰ collagen having typical spectrum characteristics,amino acid composition characteristics and peptide chain molecules of collagen protein.DSC molecular results show that the ASC and PSC are complete with three helical molecular structure,the thermal denaturation temperature(35.88 ℃,35.10℃),was significantly lower than mammals pigskin collagen denaturation temperature(41.58 ℃),which may be related to its amino acid composition differences.Collagen enzyme degradation in vitro show that the two both can be collagen enzyme degradation,enzymolysis 100 h under the condition of experiment,the enzyme degradation rate respectively(ASC:59.4%,PSC:49.4%).Using fish collagen as raw material,through the collagen molecular self-assembly fibroblasts in vitro behavior characteristics,combined with the centrifugal force,trying to build with directional structure of collagen materials.Using tensile force of materials as evaluation index,the centrifugation temperature,centrifugal time,centrifugal force and the influence of the tensile effect of mechanics of materials.The experimental results show that applying centrifugal force during collagen assemble phase can effectively improve the tensile mechanical properties of fiber materials.With the centrifugal time of 5 min,centrifugal force of 0.75 g,centrifugal temperature(25℃),the tensile ability is the best(2098KPa),significantly higher than that of contrast material(1186KPa)(p< 0.05).At the same time,the study found that in the material drying stage,along the direction of the centrifugal given continues force to exert tension(0.675 N) can further improve the tensile ability of material.On the basis of the above research,using SEM,TEM,FTIR,SAXS analysis represents the orientation microstructure of materials.SEM and TEM analysis results show that compared with contrast material,collagen fiber distribution in centrifugal directional processed materials and structure features of centrifugal force in the same direction.Ir and SAXS analysis showed that the centrifugal orientation after processing,the material orientation order of collagen increased significantly,confirmed in the assemble phase exerts a certain centrifugal force can improve the distribution of fiber assembly oriented and cause material tensile ability to significantly increase.Micro structure of collagen fibers under TEM observations showed that applying characteristic after centrifugal force function does not affect the collagen assemble D periodic structures.For oriented assembly collagen material,we further carry out the material quality and structure,thermal stability,swelling properties,resistance to enzymatic degradation performance and the performance of cell biology related research.Results show that compared with the isotropic collagen material,oriented assembly materials after PBS swelling can form lower water gel strength.Specific surface area and SEM analysis show that the oriented material had more space inside.Thermal stability analysis results show that the initial thermal denaturation of directional collagen gel temperature slightly lower than isotropic collagen gel,while enthalpy and the peak denaturation temperature is larger.The enzyme degradation performance had no significant difference,while the swelling performance in vitro of isotropic collagen is significantly lager.The cell biology of collagen material performance analysis show that compared with isotropic collagen material,oriented collagen material can better promote fibroblasts proliferation.This study showed that applying appropriate centrifugal force during collagen in vitro self-assembly stage can increase collagen fiber orientation so as to improve the tensile properties and biological performance of collagen materials.This is a simple and effective method to preparate simulation biomimetic collagen materials with good tensile. |
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